Project 4: Therapeutic Gene Editing for Rett Syndrome

项目总结项目4(Rett) Rett综合征(RTT)是一种X连锁的神经发育障碍，由MECP2基因突变引起。 大多数RTT是由相同的d8重复突变引起的，突变热点在第3和 第四外显子。这四个错义突变和四个无义突变(R106W、R133C、T158M、R168X、 R255X、R270X、R294X和R306C)占所有Rett案例的70%。RTT主要影响雌性， 发生在1-10,000名活产女婴的发病率上，并在里程碑中表现为 6-18个月大。Rett的显著特征包括逐渐丧失有目的的手部技能、言语 语言退化，步态异常，以及刻板印象的手部动作。精神障碍的其他特征 包括头部发育减速、睡眠障碍、呼吸异常和癫痫的高发生率。 RETT患者的预期寿命通常在40-50岁左右。具有里程碑意义的研究使用 有条件的MeCP2小鼠模型在症状后开启内源性MeCP2小鼠基因 令人信服地证明，与MeCP2缺失相关的神经缺陷可以逆转为显著的 学位。到目前为止，临床试验的重点是调节血管紧张素转换酶下游机制的小分子 MeCP2。有了更多的25次以上的这样的试验，仅以温和的行为证明了有限的成功 修改并且没有FDA批准的用于RTT的药物。基因和蛋白质替代策略受阻 关于需要精确滴定MECP2的受控剂量影响，因为MeCP2在 细胞是有毒的，并被疾病的X连锁性质与MECP2的细胞间变异进一步混淆 由染色体X连锁失活引起的表达。出于这些原因，基础编辑策略 直接纠正内源性基因突变和将MECP2恢复到内源性细胞水平是一种 RTT极具吸引力的治疗策略。 在这个追随者项目中，我们的目标是纠正错义和无义突变，从而能够拯救疾病 RETT患者的进展情况。具体来说，我们的目标是：(1)设计、测试和优化基本编辑、Prime编辑 和针对5个不同突变的双编辑策略；(2)在小鼠模型中测试这些策略，包括 带有工程错义和无义突变的人性化外显子。(3)进行临床前IND启用 评估安全性和有效性的研究。我们将与基因编辑核心密切合作，开发最新的基础 RTT模型系统中的编辑和/或主要编辑技术。我们将与基因编辑核心进行迭代以 确保我们的基因组编辑工具最大限度地提高目标编辑效率，最大限度地减少不必要的基因编辑 副产品和非目标编辑事件，并最大限度地与体内潜在的传递方法兼容 治疗相关性。

PROJECT SUMMARY PROJECT 4 (Rett) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene MECP2. The majority of RTT is caused by the same d eight reoccurring mutations in mutational hotspots of the 3rd and 4th exons. These four missense mutations and four nonsense mutations (R106W, R133C, T158M, R168X, R255X, R270X, R294X, and R306C) make up 70% of all Rett cases. RTT predominantly affects females, occurring at an incidence of 1-10,000 live female births and presents as a regression in milestones between the ages of 6-18 months. Hallmark characteristics of Rett include progressive loss of purposeful hand skills, speech and language regression, gait abnormalities, and stereotypic hand movements. Other features of the disorder include decelerated head growth, sleep disturbances, breathing abnormalities, and a high incidence of seizures. The life expectancy for patients with Rett is typically around 40-50 years of age. Landmark studies using conditional mouse models of Mecp2 to turn on the endogenous Mecp2 mouse gene post-symptomatically have convincingly demonstrated that neurological deficits associated with loss of Mecp2 is reversible to a significant degree. To date, clinical trials have focused on small molecules that modulate mechanisms downstream of MECP2. With more the 25+ such trials, limited success has been demonstrated with only modest behavior modifications and no approved FDA drugs for RTT. Gene and protein replacement strategies have been stymied with regards to controlled dosage affects that require precise titration of MECP2, as overexpression of Mecp2 in cells is toxic and are further confounded by the X-linked nature of the disease with cell to cell variation of MECP2 expression resulting from chromosome X-linked inactivation. For these reasons, base editing strategies that directly correct the endogenous genetic mutation and restore MECP2 to endogenous cellular levels are an extremely attractive therapeutic strategy for RTT. In this follower project, we aim to correct missense and nonsense mutations to enable rescue of disease progression in Rett patients. Specifically, we aim to: (1) Design, test and optimize Base Editing, Prime editing and Twin Editing strategies for 5 different mutations; (2) Test these strategies in mouse models containing humanized exons with engineered missense and nonsense mutations. (3) Perform pre-clinical IND enabling studies to assess safety and efficacy. We will work closely with the Gene Editing Core to develop the latest base editing and/or prime editing technologies in RTT model systems. We will iterate with the Gene Editing Core to ensure that our genome editing tools maximize on-target editing efficiencies, minimize undesirable gene editing byproducts and off-target editing events, and maximize compatibility with in vivo delivery methods of potential therapeutic relevance.